Improved thrombin inhibitors for treatment of thromboembolic conditions

Abstract

To provide thrombin inhibitors that can be used to improve ischaemic stroke outcomes, and to provide thrombin inhibitors that can be used to improve other thrombogenic disease or coagulative disorders.SOLUTION: Provided is a peptide having thrombin inhibitory activity, which comprises a specific amino acid sequence or in which a tyrosine present at a specific position is sulfated, or to provide a pharmaceutical composition comprising the peptide.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the treatment of thromboembolic conditions, fibrin clot formation and associated thrombin activity and the preparation of compounds, particularly peptides and polypeptides, for inhibiting or modifying the cleavage of fibrinogen by thrombin. [Background technology]

[0002] The reference to any prior art in this specification is not, and should not be, regarded as an admission or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction.

[0003] Ischemic stroke is caused by the development of a blood clot or embolus within the cerebral circulation and is the third leading cause of death worldwide. Stroke events are also the leading cause of disability worldwide and are associated with long, resource-intensive and costly rehabilitation programs.

[0004] Currently, the only approved pharmacological treatment for stroke to promote rapid reperfusion of the ischemic brain, thereby minimizing stroke events, is the intravenous (iv) delivery of the thrombolytic agent, recombinant tissue plasminogen activator (rtPA), which activates plasminogen to plasmin, which then degrades fibrin and other clot-binding proteins, thereby improving blood flow through affected blood vessels.

[0005] Despite its widespread clinical use, rtPA-based therapy has several limitations in both efficacy and application. Of particular concern is that only 20-30% of patients experience complete arterial recanalization after rtPA therapy, and 20-30% of these patients will experience reocclusion. This problem is thought to be due to clot-bound thrombin retaining activity for cleaving fibrinogen to fibrin, since plasmin induced by rtPA degrades fibrin in clots.

[0006] Of further concern is the observed increased incidence of intracranial hemorrhage (ICH) with rtPA therapy, which substantially limits the dose of rtPA that can be given for thrombolytic therapy.

[0007] The significant limitations of rtPA therapy have sparked renewed interest in the development of improved thrombolytic therapies.

[0008] Thrombin plays a central role in clot formation, primarily through the generation of insoluble fibrin, and therefore thrombin inhibitors have emerged as promising candidates for use as adjunctive therapy alongside rtPA.

[0009] To date, the indirect thrombin inhibitor heparin and the direct thrombin inhibitors (DTIs) hirudin and argatroban have been studied. Although there has been an overall improvement in revascularization, there is an increased risk of bleeding and symptomatic ICH associated with these combination therapies. Summary of the Invention [Problem to be solved by the invention]

[0010] There is a need for thrombin inhibitors that can be used to improve the prognosis of ischemic stroke.

[0011] Thoronamine can be used in combination with rtPA to improve the outcome of ischemic stroke There is also a need for bin inhibitors.

[0012] There is also a need for thrombin inhibitors that can be used to ameliorate other thrombogenic or coagulation disorders. [Means for solving the problem]

[0013] The present invention seeks to address one or more of the above mentioned needs or limitations and in one embodiment provides a method for the preparation of a nucleic acid sequence comprising SEQ ID NO:1: YPESDSAKDDGNQEKEKALLVKVQERSDDGDYDEYDNDETTHTPDPSAPTARPRIREHQA The present invention provides a peptide comprising, consisting essentially of, or consisting of the amino acid sequence shown in

[0014] In another embodiment, the present invention relates to a nucleic acid sequence similar to SEQ ID NO:2: YPERDSAKDGNEEQERALPVNVQERGEVADADYDDYDEEGTPTVDPTAQTARPRLQGNQS The present invention provides a peptide comprising, consisting essentially of, or consisting of the amino acid sequence shown in

[0015] Preferably, the peptide consists of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. More preferably, the peptide consists of the amino acid sequence shown in SEQ ID NO: 1.

[0016] In another embodiment, the peptide comprises, consists essentially of, or consists of a biologically active fragment or variant of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0017] The peptides can be recombinant, isolated or synthetic peptides.

[0018] In either embodiment, the peptide at least partially inhibits thrombin.

[0019] Typically, at least one residue of the peptide is a sulfated tyrosine or a sulfated tyrosine mimetic residue, preferably a sulfated tyrosine residue.

[0020] Preferably, the tyrosine at position 32 in SEQ ID NO:1 is sulfated and the tyrosine at position 35 is sulfated.

[0021] In another embodiment, the tyrosine at position 32 in SEQ ID NO:1 is sulfated. The tyrosine at position 35 in SEQ ID NO:1 can be non-sulfated.

[0022] In another embodiment, the tyrosine at position 35 in SEQ ID NO:1 is sulfated. The tyrosine at position 32 in SEQ ID NO:1 can be non-sulfated.

[0023] Preferably, the tyrosine at position 33 in SEQ ID NO:2 is sulfated and the tyrosine at position 36 is sulfated.

[0024] In another embodiment, the tyrosine at position 33 in SEQ ID NO:2 is sulfated. The tyrosine at position 36 in SEQ ID NO:2 can be non-sulfated.

[0025] In another embodiment, the tyrosine at position 36 in SEQ ID NO:2 is sulfated. The tyrosine at position 33 in SEQ ID NO:2 may be non-sulfated.

[0026] In another embodiment, Formula 1: A.B. A fusion peptide of the formula: A is a peptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 and comprising one or more of the properties described above; B is SEQ ID NO:3: DFEEIPEEYLQ or SEQ ID NO:4: DEDYAAIEASLSETF or SEQ ID NO:5: PFDFEAIPEEYLDDES A peptide comprising, consisting essentially of, or consisting of the amino acid sequence shown in

[0027] Preferably, B binds to exosite I of thrombin.

[0028] Region A can be located N-terminal to B. In another embodiment, B is located N-terminal to A.

[0029] In one embodiment, A and B may be directly linked. In another embodiment, A and B may include a linker.

[0030] In any embodiment, the peptides of the invention at least partially inhibit thrombin. Preferably, the peptides of the invention at least partially inhibit thrombin activity by at least 1%, 5%, 10%, 25%, 50%, 60%, 70%, 80%, or 90% or more. Preferably, the peptides of the invention inhibit thrombin with an inhibition constant K of less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 500 pM, or less than about 200 pM.

[0031] In one embodiment, the peptides of the invention reduce clotting time by at least 20% based on molar equivalents of peptide and known thrombin inhibitor, as compared to known thrombin inhibitors that result in significant blood loss, as measured using the in vitro activated partial thromboplastin time (aPTT) assay described herein. Preferably, the known thrombin inhibitor is hirudin.

[0032] In one embodiment, the peptides of the invention comprise a clotting time of about 60 seconds to about 200 seconds at a concentration of greater than about 4 μg / mL, as measured using an in vitro activated partial thromboplastin time (aPTT) assay.

[0033] In one embodiment, the peptides of the invention provide a clotting time of about 60 seconds to about 200 seconds when administered to an individual in an amount of about 1 μg per kg of the individual to about 10 mg per kg of the individual, as measured ex vivo using an in vitro activated partial thromboplastin time (aPTT) assay.

[0034] In one embodiment, the peptides of the present invention do not result in significant blood loss. Preferably, the peptides do not result in significant blood loss as measured using any assay capable of determining blood loss. More preferably, the peptides, when administered to an individual, reduce blood loss by at least 10-fold based on molar equivalents of the peptide and the known thrombin inhibitor, compared to known thrombin inhibitors that result in significant blood loss. Preferably, the peptides reduce blood loss by at least 10-fold based on molar equivalents of the peptide and the known thrombin inhibitor, compared to known thrombin inhibitors that result in significant blood loss. The bin inhibitor is hirudin or argatroban, more preferably hirudin.

[0035] In one embodiment, the peptides of the invention, when administered to an individual, reduce symptomatic intracerebral hemorrhage (sICH) as compared to hirudin based on the molar equivalent of the peptide and hirudin.

[0036] In another embodiment, there is provided a polynucleotide, preferably a cDNA, comprising a nucleotide sequence encoding a peptide comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 as generally described above, or a peptide of Formula 1 as described above, as well as vectors, expression constructs and cells containing said polynucleotides.

[0037] Preferably, the cells contain a sulfotransferase that allows for the sulfation of tyrosine.

[0038] In one embodiment, sulfotyrosine is genetically incorporated into a peptide according to the invention by amber codon suppression.

[0039] In another embodiment, there is provided a pharmaceutical composition comprising a peptide comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 as generally described above or a peptide of formula 1 as described above, and a pharma- ceutically effective carrier, diluent or excipient.

[0040] The present invention also provides a method of preventing or treating a thromboembolic condition in an individual in need thereof, comprising, consisting essentially of, or consisting of administering to the individual a therapeutically effective amount of a peptide of the invention, thereby preventing or treating the thromboembolic condition in the individual.

[0041] The present invention also provides a method of treating a thromboembolic condition or reducing the severity of symptoms of a thromboembolic condition in an individual, comprising, consisting essentially of, or consisting of administering to an individual in need thereof a therapeutically effective amount of a peptide of the invention, thereby treating the thromboembolic condition or reducing the severity of symptoms of a thromboembolic condition in the individual.

[0042] In another aspect, the invention provides a method for inhibiting or minimizing the progression of symptoms of a thromboembolic condition in an individual comprising, consisting essentially of, or consisting of administering a therapeutically effective amount of a peptide of the invention to an individual experiencing symptoms of a thromboembolic condition, thereby inhibiting or minimizing the progression of symptoms of the thromboembolic condition in the individual.

[0043] Thus, in a further aspect, the present invention provides a method of reducing the severity of a thromboembolic condition in an individual in need thereof, comprising, consisting essentially of or consisting of the step of administering to the individual in need thereof a therapeutically effective amount of a peptide of the invention, thereby reducing the severity of the thromboembolic condition in the individual.

[0044] In another aspect, the present invention provides a method for inhibiting or minimizing the progression of blood clot formation associated with inflammation caused by viral infection in an individual, comprising, consisting essentially of, or consisting of administering a therapeutically effective amount of a peptide of the present invention to an individual experiencing inflammation caused by viral infection, thereby inhibiting or minimizing the progression of blood clot formation in the individual.

[0045] Thus, in a further aspect, the present invention relates to a method for treating inflammation caused by a viral infection. The present invention provides a method for reducing the severity of blood clot formation associated with a pulmonary artery disease in an individual in need thereof, comprising, consisting essentially of, or consisting of, administering to the individual in need thereof a therapeutically effective amount of a peptide of the present invention, thereby reducing the severity of blood clot formation in the individual.

[0046] In another aspect, the present invention provides a method for producing a composition comprising: · preventing or treating a thromboembolic condition in an individual; Treating a thromboembolic condition or reducing the severity of the symptoms of a thromboembolic condition in an individual; inhibiting or minimizing the progression of symptoms of a thromboembolic condition in an individual; and / or · Reducing the severity of thromboembolic conditions in individuals; Inhibiting or minimizing the progression of blood clot formation associated with inflammation caused by a viral infection in an individual; and / or Reducing the severity of blood clot formation associated with inflammation caused by viral infections There is also provided the use of a peptide of the invention in the manufacture of a medicament for

[0047] In another aspect, the present invention provides a method for producing a composition comprising: · preventing or treating a thromboembolic condition in an individual; Treating a thromboembolic condition or reducing the severity of the symptoms of a thromboembolic condition in an individual; - inhibiting or minimizing the progression of symptoms of a thromboembolic condition in an individual; · Reducing the severity of thromboembolic conditions in individuals; Inhibiting or minimizing the progression of blood clot formation associated with inflammation caused by a viral infection in an individual; and / or Reducing the severity of blood clot formation associated with inflammation caused by viral infections Also provided is a peptide of the invention for use in

[0048] In another aspect, the present invention provides a method for producing a composition comprising: · preventing or treating a thromboembolic condition in an individual; Treating a thromboembolic condition or reducing the severity of the symptoms of a thromboembolic condition in an individual; - inhibiting or minimizing the progression of symptoms of a thromboembolic condition in an individual; · Reducing the severity of thromboembolic conditions in individuals; Inhibiting or minimizing the progression of blood clot formation associated with inflammation caused by a viral infection in an individual; and / or Reducing the severity of blood clot formation associated with inflammation caused by viral infections Also provided is a pharmaceutical composition comprising, consisting essentially of, or consisting of a peptide of the invention and a pharma- ceutically acceptable carrier, diluent or excipient for use in.

[0049] In another aspect, the present invention provides a pharmaceutical composition for preventing or treating a thromboembolic condition in an individual comprising a peptide of the present invention as an active ingredient and a pharma- ceutical acceptable diluent, excipient or carrier, hi one embodiment, the only active ingredient present in the composition is the peptide of the present invention.

[0050] In another aspect, the present invention provides a pharmaceutical composition for preventing or treating a thromboembolic condition in an individual comprising a peptide of the present invention as a main ingredient and a pharma- ceutical acceptable diluent, excipient or carrier, hi one embodiment, the only active ingredient present in the composition is the peptide of the present invention.

[0051] In any of the methods, uses or compositions of the invention described above, the only active ingredient present in the composition is the peptide of the invention.

[0052] Alternatively, in any of the methods, uses or compositions of the invention described above, the composition may optionally comprise a further active ingredient, preferably selected from rtPA therapy, a platelet inhibitor or a combination thereof.

[0053] In any of the methods, uses or compositions of the invention described above, the peptide of the invention can be administered in combination with one or more active agents. Preferably, the one or more active agents are selected from rtPA therapy, platelet inhibitors or a combination thereof. The administration of the peptide of the invention and the active agent can include simultaneous, sequential and / or separate (immediate or long-delayed) administration. The sequential and / or separate administration can be in any order. For example, the peptide of the invention can be administered to an individual before or after the administration of the active agent. Preferably, when the active agent is rtPA therapy, the peptide is administered after the administration of the active agent.

[0054] In any of the methods or uses of the invention described herein, the peptides of the invention may be administered parenterally, preferably intravenously.

[0055] In another aspect, the present invention provides a kit for use in or, when used, in the method of the invention, comprising: a peptide of the invention; and optionally - written instructions describing the use of the peptide of the invention in the method of the invention; Also provided is a kit comprising, consisting essentially of, or consisting of:

[0056] As used herein, unless otherwise required by context, the term "comprise" and variations of this term, such as "comprises", "comprises" and "comprised", are not intended to exclude additional additives, components, integers or steps.

[0057] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example, and with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0058] [Figure 1] Inhibition of human thrombin by differently sulfated peptides of SEQ ID NO:1 (MDL-2) analogues; Un (unsulfated), Y32 (sulfated at Y32), Y35 (sulfated at Y35), DS (sulfated at Y32 and Y35). (A) Raw data; (B) Table of inhibition constants (Ki). [Diagram 2] Inhibition of human thrombin by differently sulfated peptides of SEQ ID NO:2 (MDL-1) analog; Un (unsulfated), Y33 (sulfated at Y33), Y36 (sulfated at Y36), DS (sulfated at Y33 and Y36). (A) Raw data; (B) Table of inhibition constants (Ki). [Diagram 3] Comparative anticoagulation and bleeding profiles of hirudin, argatroban and sequence number 1 (MDL-2): (A) Activated partial thromboplastin time (aPTT) dose-response curves in mouse plasma comparing hirudin (n=5), argatroban (n=3) and MDL-2 (n=5); (B) MDL-2 clotting activity in vivo over time; (C) MDL-2 human plasma stability over time. [Figure 4] Effect of hirudin, argatroban and SEQ ID NO: 1 (MDL-2) on mouse tail bleeding time. [Diagram 5] (A) Schematic of experimental thrombolysis-rtPA / anticoagulant combination and vessel recanalization. Carstair's histological staining was performed on excised carotid arteries from sham control mice (uninjured vessel) (B) or excised carotid arteries 10 min (C-D) or 1 h (E) after carotid artery occlusion. Staining reveals platelets (blue / purple), fibrin (crimson / purple) and red blood cells (yellow / orange) (5 μm sections, paraffin-embedded tissue). Collagen of the vessel wall is shown in blue surrounding the thrombus. [Figure 6]rtPA / anticoagulant combination and vascular recanalization: (A) no treatment; (B) rtPA (10 mg / kg); (C) rtPA (10 mg / kg) and hirudin (1 mg / kg); (D) rtPA (10 mg / kg) and SEQ ID NO:1 (MDL-2) (10 mg / kg). [Figure 7] Analytical HPLC trace (A) and mass spectrum (B) of purified SEQ ID NO:1 (MadL2(1-30) selenoester. [Figure 8] Analytical HPLC trace (A) and mass spectrum (B) of purified SEQ ID NO:1 (MadL2) DS(31-60) diselenide. [Figure 9] Analytical HPLC traces (0-50% B over 5 min, 0.1% v / v TFA, λ=214 nm) and mass spectra of (A) crude ligation, (B) crude deselenation, (C) crude neopentyl ester deprotection, and (D) purified SEQ ID NO:1(MadL2)DS. [Figure 10] MALDI-TOF mass spectra of sequence number 1 (MadL2) DS (A) 4000-20000 m / z and (B) 6600-7400 m / z. [Figure 11] Analytical HPLC trace (A) and mass spectrum (B) of purified SEQ ID NO:2 (MadL1)(1-28) selenoester. [Figure 12] Analytical HPLC trace (A) and mass spectrum (B) of purified SEQ ID NO:2 (MadL1) DS(29-61) diselenide. [Figure 13] Analytical HPLC traces (0-50% B over 30 min, 0.1% v / v TFA, λ=214 nm) and mass spectra of (A) crude ligation, (B) crude deselenation, (C) crude neopentyl ester deprotection, and (D) purified SEQ ID NO:2(MadL1)DS. [Figure 14] MALDI-TOF mass spectra of sequence number 2 (MadL1) DS (A) 4000-22000 m / z and (B) 6800-7300 m / z. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] It will be understood that the invention disclosed and defined herein extends to all other combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of which different combinations constitute various alternative aspects of the invention.

[0060] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intent is not to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the present invention as defined by the claims.

[0061] Disclosed herein are peptides that inhibit thrombin. The inventors have surprisingly discovered that the thrombin inhibitor peptides disclosed herein do not induce substantial blood loss compared to other known thrombin inhibitors, making them useful for preventing, treating or slowing the progression of conditions associated with thrombin generation and / or action. For example, the thrombin inhibitor peptides of the present invention are useful for preventing, treating or slowing the progression of thromboembolic conditions. In a particularly preferred embodiment, the thromboembolic condition is stroke. The thrombin inhibitor peptides of the present invention may be useful for treating stroke, especially when combined with rtPA therapy, optionally with a platelet inhibitor.

[0062] To inhibit fibrin formation by clot-bound thrombin, these thrombin inhibitors It is proposed that therapeutic peptides may be utilized to address some of the recanalization problems associated with rtPA therapy. Thrombin inhibitor peptides may also be used to alter the binding affinity and / or function of other peptides that bind to the thrombin active site and / or thrombin exosite and block function.

[0063] General Throughout this specification, unless specifically stated otherwise or unless the context requires otherwise, references to a single step, material composition, group of steps or group of material compositions shall be construed to include one and a plurality (i.e., one or more) of those steps, material compositions, group of steps or group of material compositions. Thus, as used herein, the singular forms "a," "an," and "the" include plural aspects and vice versa, unless the context clearly dictates otherwise. For example, references to "a" include the single and the more; references to "an" include the single and the more; references to "the" include the single and the more; etc.

[0064] Those skilled in the art will appreciate that the present invention is capable of variations and modifications other than those specifically described. It will be understood that the present invention includes all such variations and modifications. The present invention includes, individually or collectively, all of the steps, features, compositions and compounds referred to or shown in this specification, as well as any and all combinations or any two or more of said steps and features.

[0065] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention, and the present invention is in no way limited to the methods and materials described.

[0066] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0067] The present invention is not to be limited in scope by the specific examples described herein, which are merely illustrative. Functionally equivalent products, compositions and methods are clearly within the scope of the invention.

[0068] Any example or embodiment of the invention herein is to be construed as applying mutatis mutandis to any other example or embodiment of the invention, unless specifically stated otherwise.

[0069] Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in the fields of cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0070] The term "and / or", e.g., "X and / or Y", shall be understood to mean either "X and Y" or "X or Y" and shall be interpreted as providing clear support for both meanings or for either meaning.

[0071] Use of the term "about" includes and refers to the value or parameter itself. For example, "about x" includes and refers to "x" itself. In some embodiments, the term "about" when used in reference to a measurement or to modify a value, unit, constant, or range of values, refers to a variation of ±10%. For example, in some embodiments, "about 60" includes 54 to 66.

[0072] Select Definition "Thrombin" is a serine protease that has a central role in hemostasis through the conversion of fibrinogen to fibrin.

[0073] The "thrombin active site" is the catalytic site that cleaves a variety of substrates, including fibrinogen, fibrinopeptides, factor V, factor VIII, protease-activated receptors (PARs), glycoprotein V, factor XI, factor XIII, ADAMTS13, and protein C.

[0074] The "thrombin exosite II" (also known as the "heparin-binding exosite") is a positively charged recognition surface that affects substrate and cofactor binding, specifically fibrinogen, Gp1bα and heparin. It can include residues R93, R101, R126, K236, K240 and R233.

[0075] "Thrombin exosite I" (also known as the "fibrinogen-binding exosite") is a positively charged recognition surface that affects substrate and cofactor binding, specifically fibrinogen, factor V, factor VIII, ADAMTS13, factor XIII, PAR-1, factor XI and thrombomodulin. It can include residues K36, H71, R73, R75, Y76 and R77.

[0076] Thrombin is divided into lanes 1 and 2, including the thrombin active site and exosites I and II. D. et al. 2005 Blood J. 106:2605-2612.

[0077] "Sulfated tyrosine residue", "tyrosine-O-sulfate", "sTyr" and "TyrSO3" are residues resulting from the transfer of a sulfur-containing group to the hydroxyl side chain of tyrosine. This can be generated by the action of protein tyrosine sulfotransferase (TPST).

[0078] "Sulfated tyrosine mimetics" refers to mimetics of sulfated tyrosine residues, including, but not limited to, tyrosine sulfonate and tyrosine difluorosulfonate.

[0079] The terms "thrombin inhibitors" and "anticoagulants" are used interchangeably herein.

[0080] It will be understood that the phrase "inhibits thrombin" means that the peptide of the present invention inhibits or reduces thrombin activity. Thrombin activity refers to the ability of thrombin to convert fibrinogen to fibrin. Moreover, the activity is measured using a suitable in vitro, cellular or in vivo assay, and the thrombin activity is inhibited or reduced by at least 1%, 5%, 10%, 25%, 50%, 60%, 70%, 80% or 90% or more compared to the thrombin activity in the same assay under the same conditions without the peptide.

[0081] "Clotting time" generally refers to the time required for the formation of a fibrin clot. Typically, clotting time is assessed by the activated partial thromboplastin time (aPTT) test.

[0082] "Blood loss" generally refers to excessive bleeding or blood loss. Minor blood loss refers to bleeding that is not clinically significant. Significant blood loss refers to internal bleeding or when an individual is experiencing hemodynamic instability. Blood loss refers to the loss of sufficient blood to cause cataracts. Blood loss can be measured clinically by measuring blood pressure, pulse rate, hemoglobin levels, urea levels in blood, and / or by imaging. Indicators of significant blood loss will be apparent to those skilled in the art. Blood loss can also be determined experimentally, for example, by measuring the total volume of blood collected from a mouse tail cut, and can be quantified by measuring the hemoglobin (mg / dL) present in the total volume of blood lost during a specified observation period (e.g., 20 minutes). Total hemoglobin can be assessed by a commercially available hemoglobin colorimetric assay kit (catalog number MAK115; Sigma-Aldrich).

[0083] As used herein, the phrase "significant blood loss" shall be understood to mean excessive blood loss and shall mean an increase in the amount of hemoglobin of at least about 5-fold, 10-fold, 20-fold, 50-fold or more compared to the amount of hemoglobin in the same assay under the same conditions without the active agent. As used herein, the phrase "does not induce significant blood loss" shall be understood to mean an amount of hemoglobin that is about 5-fold, 4-fold, 3-fold, 2-fold or less than the amount of hemoglobin in the same assay under the same conditions without the active agent.

[0084] As used herein, the phrase "reducing blood loss" may be understood to mean a reduction in the amount of hemoglobin by at least about 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold or more based on the molar equivalent of the peptide and the known thrombin inhibitor, compared to known thrombin inhibitors that result in significant blood loss when administered to an individual.

[0085] Examples of "known thrombin inhibitors" that induce blood loss include vitamin K antagonists (or coumarin-based anticoagulants), low molecular weight heparins (LMWHs), direct thrombin inhibitors (DTIs) or factor Xa inhibitors. LMWHs include, for example, bemiparin, certoparin, dalteparin, enoxaparin, nadroparin, parnaparin, reviparin and tinzaparin. DTIs include, for example, lepirudin, desirudin, bivalirudin, argatroban, dabigatran and antithrombin III. Factor Xa inhibitors include, for example, apixaban, fondaparinux, rivaroxaban and edoxaban. In one example, a known thrombin inhibitor is hirudin. "Hirudin" is a low molecular weight peptide (7 kDa) consisting of 65 amino acids (Dodt et al., 2002). al., 1984 FEBS Lett., 165:180-4) and prevents blood from clotting by binding to exosite I and the thrombin active site (Stone and Hofsteenge, 1986 Biochem, 25:4622-28).

[0086] As used herein, the term "condition" refers to a disruption or interruption of normal function and is not to be limited to any particular condition and includes a disease or disorder.

[0087] As used herein, the terms "preventing," "prevent" or "prevention" include administering a peptide of the invention to stop or slow the onset of at least one symptom of a condition. The terms also encompass treatment of a subject in remission to prevent or slow recurrence.

[0088] As used herein, the terms "treating," "treat" or "treatment" include administering a peptide described herein to thereby reduce or eliminate at least one symptom of a particular condition.

[0089] The phrase "therapeutically effective amount" generally refers to an amount of one or more peptides of the invention that (i) treats a particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. , an amount sufficient to prolong in vitro clotting time by 2-3 fold or more depending on the therapeutic indication, an amount effective to decrease thrombin generation or directly inhibit thrombin activity.

[0090] As used herein, the term "individual" shall be taken to mean any animal, e.g., mammal, including humans. Exemplary individuals include, but are not limited to, humans and non-human primates. For example, the individual is a human. The present invention finds particular application in humans, but is also useful for veterinary therapeutic purposes. The present invention is useful for farm animals or livestock, e.g., cows, sheep, horses, and poultry; companion animals, e.g., cats and dogs; and zoo animals.

[0091] "Peptide" as used herein refers to a polymer of amino acid residues and to the variants and synthetic analogues of polymers of amino acid residues.Thus, this term applies to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as chemical analogues of corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers.This term does not exclude modifications to amino acid side chains or N- or C-terminus of peptide.

[0092] The thrombin inhibitor peptides described herein can have at least one conservative substitution of an amino acid residue. Preferably, this conservative substitution does not change the overall conformation or function of the peptide. Preferably, a conservative substitution involves replacing one amino acid with another that has one or more similar properties.

[0093] Amino acids with similar properties are well known in the art.For example, polar / hydrophilic amino acids that can be replaced include asparagine, glutamine, serine, threonine, lysine, arginine, histidine, aspartic acid and glutamic acid; non-polar / hydrophobic amino acids that can be replaced include glycine, alanine, valine, leucine, isoleucine, proline, tyrosine, phenylalanine, tryptophan and methionine; acidic amino acids that can be replaced include aspartic acid and glutamic acid, and basic amino acids that can be replaced include histidine, lysine and arginine.

[0094] The peptides described herein can incorporate non-natural or unnatural amino acids. Any amino acid can be natural or unnatural / unconventional unless otherwise specified. Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, the use of norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or D-isomers of amino acids.

[0095] As used herein, "MDL-2," "MadL-2," and "madanin-like 2" refer to the amino acid sequence as set forth in SEQ ID NO:1.

[0096] As used herein, "MDL-1," "MadL-1," and "madanin-like 1" refer to the amino acid sequence as set forth in SEQ ID NO:2.

[0097] Thrombin Inhibitor Peptides In one embodiment, SEQ ID NO:1: YPESDSAKDDGNQEKEKALLVKVQERSDDGDYDEYDNDETTHTPDPSAPTARPRIREHQA A peptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in Provided.

[0098] In another embodiment, SEQ ID NO:2: YPERDSAKDGNEEQERALPVNVQERGEVADADYDDYDEEGTPTVDPTAQTARPRLQGNQS In accordance with the present invention, there is provided a peptide comprising, consisting essentially of, or consisting of the amino acid sequence shown in

[0099] Preferably, the peptide consists of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. More preferably, the peptide consists of the amino acid sequence shown in SEQ ID NO: 1.

[0100] The peptide may comprise, consist essentially of, or consist of a biologically active fragment or variant of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0101] As used herein, a "biologically active fragment" refers to a portion or subsequence of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, including a domain thereof having 10% or more, preferably 25% or more, more preferably 50% or more, and even more preferably 75%, 80%, 85%, 90% or 95% or more of the biological activity of a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. Such activity can generally be assessed using standard testing methods and bioassays that can be recognized by those skilled in the art as useful for determining such activity.

[0102] A fragment of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 can constitute fewer than 60, fewer than 50, fewer than 40, fewer than 30, fewer than 20, or fewer than 10 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. Multiple fragments of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 are also contemplated.

[0103] "Domain" (of a protein) means a portion of a protein sharing common structural, physiochemical and functional features (e.g., hydrophobic, polar, globular or helical domains) or properties (e.g., a protein-binding domain, such as an exosite II binding domain or a thrombin active site domain, a receptor binding domain, a cofactor binding domain, etc.).

[0104] Also contemplated are variants of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 that contain one or more amino acid substitutions, insertions and / or deletions within SEQ ID NO:1 (or a fragment thereof) or SEQ ID NO:2 (or a fragment thereof) compared to the wild-type SEQ ID NO:1 / SEQ ID NO:2.

[0105] Typically, with respect to proteins, a "variant" protein also has one or more amino acids replaced by different amino acids. It is well understood that an amino acid can be changed to another amino acid with broadly similar properties without changing the nature of the protein's activity (i.e., conservative substitutions).

[0106] It will also be understood that one or more amino acid residues of a reference sequence such as SEQ ID NO:1 / SEQ ID NO:2 (or a fragment thereof) can be modified or deleted, or additional sequences added, without substantially altering the biological activity of the amino acid sequence shown in SEQ ID NO:1 / SEQ ID NO:2 (or a fragment thereof). Such activity can generally be assessed using standard testing methods and bioassays that can be recognized by the skilled artisan as useful for determining such activity.

[0107] In one embodiment, the protein variant comprises a polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:2. It shares at least 80%, preferably at least 85%, more preferably at least 90%, 95%, 98% or 99% sequence identity with the subsequence.

[0108] Preferably, sequence identity is measured over at least 60%, more preferably at least 75%, more preferably at least 90% or more preferably at least 95%, 98% or substantially the full length of a reference sequence.

[0109] To determine the sequence identity (percentage), optimal alignment of amino acid and / or nucleotide sequences can be performed by running computer algorithms (GAP, BESTFIT, FASTA and TFASTA in the Geneworks program by Intelligenetics; Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, WI, USA) or by searching and finding the best alignment (i.e. resulting in the highest percentage of homology over the comparison window) produced by any of the various methods selected. Reference can also be made to the BLAST family of programs, as disclosed, for example, by Altschul et al., Nucl. Acids Res. 25:3389-402, 1997.

[0110] A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

[0111] The peptides at least partially inhibit thrombin, and thus may remove or reduce clot-bound thrombin from clots in subjects of rtPA thrombolytic therapy, thereby minimizing the occurrence of reduced recanalization otherwise associated with rtPA therapy.

[0112] Typically, at least one residue of the peptide is a sulfated tyrosine residue or a sulfated tyrosine mimetic residue, preferably a sulfated tyrosine residue.

[0113] Preferably, the tyrosine at position 32 in SEQ ID NO:1 is sulfated and the tyrosine at position 35 is sulfated.

[0114] In another embodiment, the tyrosine at position 32 in SEQ ID NO:1 is sulfated. The tyrosine at position 35 in SEQ ID NO:1 can be non-sulfated.

[0115] In another embodiment, the tyrosine at position 35 in SEQ ID NO:1 is sulfated. The tyrosine at position 32 in SEQ ID NO:1 can be non-sulfated.

[0116] Preferably, the tyrosine at position 33 in SEQ ID NO:2 is sulfated and the tyrosine at position 36 is sulfated.

[0117] In another embodiment, the tyrosine at position 33 in SEQ ID NO:2 is sulfated. The tyrosine at position 36 in SEQ ID NO:2 can be non-sulfated.

[0118] In another embodiment, the tyrosine at position 36 in SEQ ID NO:2 is sulfated. The tyrosine at position 33 in SEQ ID NO:2 may be non-sulfated.

[0119] As described in the Examples herein, sulfation of one or both tyrosine residues enhances the thrombin synthesis. This is associated with improved inhibition of agonist activity.

[0120] The peptides can be recombinant, isolated or synthetic peptides.

[0121] In one embodiment, sulfation of tyrosine residues can be achieved by expressing a nucleotide sequence encoding a peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 in a cell containing a protein tyrosine sulfotransferase (TPST). More specifically, inorganic sulfate can be activated in the form of adenosine-5'-phosphosulfate (APS) and 3'-phosphoadenosine-5'-phosphosulfate (PAPS) by ATP sulfurylase and APS kinase, respectively. The activated sulfate can then be transferred to tyrosine by TPST in the Golgi apparatus.

[0122] Another approach to obtain sulfated tyrosine residues in the peptides of the present invention is to utilize a recombinant expression system that contains amber codon suppression that allows sulfotyrosine to be incorporated into the peptide during its recombinant synthesis (Liu, CC and Schultz PG, Nature Biotechnology 2006; Italia JS et al Nature Chemical Biology 2020).

[0123] In the Examples herein, we provide a synthetic methodology that allows for the generation of homogeneous compositions of sulfated peptides (i.e., compositions containing peptides with only one sulfation profile).

[0124] Thrombin Inhibitor Fusion Peptides In a further embodiment, the present invention relates to utilizing the thrombin inhibitor peptides disclosed herein for the design, modification and / or generation of novel inhibitors of thrombin activity, in particular inhibitors to prevent or at least minimize cleavage of fibrinogen or fibrinopeptides by thrombin. These thrombin inhibitor peptides can provide inhibitors with improved affinity for binding to exosite I.

[0125] The thrombin inhibitor fusion peptide has the formula 1: A.B. where: A is a peptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 and having one or more of the properties described above; B is SEQ ID NO:3: DFEEIPEEYLQ or SEQ ID NO:4: DEDYAAIEASLSETF or SEQ ID NO:5: PFDFEAIPEEYLDDES A peptide comprising, consisting essentially of, or consisting of the amino acid sequence shown in

[0126] Preferably, B binds to exosite I of thrombin.

[0127] Region A can be located N-terminal to B. In another embodiment, B is located N-terminal to A.

[0128] In one embodiment, A and B may be directly linked.

[0129] In another embodiment, A and B can include a linker. The thrombin inhibitor fusion peptide of formula 1 can include a linker in the form of a peptide sequence (e.g., a peptide containing two or more amino acid residues, such as Gly and Ala) or other polymer (e.g., diethylene glycol linker) that connects B() with A. Examples of peptide sequences include polyAla or polyGly peptides. The length of the linker peptide can be determined according to the molecular distance between different sites on thrombin.

[0130] Peptide synthesis The peptides and fusion peptides described above can be prepared by solid-phase peptide synthesis. For example, the fusion peptide of formula 1 can be prepared by a method that includes the steps of solid-phase synthesis of A, solid-phase synthesis of B and linking A to B or alternatively linking A to a linker and linking the A-linker complex to B in the final step to form an A-linker-B complex. Alternatively, AB can be prepared by a single solid-phase peptide synthesis without linking.

[0131] In other embodiments, the peptides can be synthesized by recombinant DNA technology. It is particularly preferred that the cell line used in this technology is capable of (i) growing in the presence of inorganic sulfate and (ii) assimilating inorganic sulfate into a biological system, particularly a system that involves post-translational modification of tyrosine residues. Such cell lines generally contain a protein tyrosine sulfotransferase in the Golgi apparatus that allows the formation of one or more tyrosine-O-sulfate residues. In certain embodiments, the expression product can be heterogeneous with respect to the tyrosine sulfation pattern. A homogeneous population of tyrosine-sulfated isoforms can be obtained by purifying the expression product on various separation systems, including chromatographic systems that allow the differentiation of isoforms based on tyrosine-sulfated phenotype.

[0132] Assay of peptide activity Binding to thrombin The peptides described herein can be tested for thrombin selectivity by counterscreening against a panel of proteases from the blood coagulation cascade including trypsin, chymotrypsin, elastase, papain, reptilase, and factor Xa and activated protein C. Inhibitors are initially screened at a single concentration (5 μM) using a fluorescence polarization assay as described above.

[0133] The molecular details of thrombin recognition and inhibition by the inhibitors described herein can be determined by solving the three-dimensional structure of their complex with thrombin. Briefly, thrombin-inhibitor complexes are prepared in vitro and subjected to extensive submicroliter scale screening of crystallization conditions. Preliminary conditions are refined and optimized using custom grid screens. Cryoprotection conditions are determined and initial sample characterization is performed using an X-ray diffractometer. High-resolution X-ray diffraction data are collected at a high-brilliance synchrotron source to ensure adequate detail in the resulting model. The structure is solved by molecular replacement techniques using the coordinates of unliganded human thrombin as a search model, and refined and interpreted using a computational platform. These data provide details about the inhibitor's binding mechanism and reveal key interactions with thrombin.

[0134] Determination of inhibitory activity The thrombin inhibitory activity of the peptides was determined spectrophotometrically by measuring the inhibition of the amidolytic activity of human α-thrombin using Tos-Gly-Pro-Arg-p-nitroanilide as a chromogenic substrate. The inhibition rate can be determined by continuously measuring the inhibition rate. Inhibition assays can be performed using 0.2 nM enzyme, 100 μM substrate and increasing concentrations of peptide. The concentration of peptide can be determined using a Direct Detect infrared spectrometer. The inhibition constant (Ki) of the peptide can be determined according to the tight binding model by fitting the steady-state kinetic inhibition data to the Morrison equation. The reaction progress can be monitored for 1-2 hours at 405 nm on a microplate reader. Dose-response curves can be used to determine the Ki value.

[0135] Preferably, the peptides of the invention at least partially inhibit thrombin activity by at least 1%, 5%, 10%, 25%, 50%, 60%, 70%, 80% or 90% or more.

[0136] Preferably, the peptides of the invention inhibit thrombin with an inhibition constant K of less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 500 pM, less than about 200 pM. Preferably, a peptide comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in SEQ ID NO:1, in which at least one residue of the peptide is a sulfated tyrosine residue, inhibits thrombin with an inhibition constant K of less than about 3 nM. More preferably, a peptide comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in SEQ ID NO:1, in which the tyrosine at position 32 and the tyrosine at position 35 in SEQ ID NO:1 are sulfated, inhibits thrombin with an inhibition constant K of less than about 200 pM, preferably less than about 160 pM. Preferably, a peptide comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in SEQ ID NO:2, in which at least one residue of the peptide is a sulfated tyrosine residue, inhibits thrombin with an inhibition constant Ki of less than about 20 nM. More preferably, a peptide comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in SEQ ID NO:2, in which the tyrosine at position 33 in SEQ ID NO:2 is sulfated, inhibits thrombin with an inhibition constant Ki of less than about 4 nM. More preferably, a peptide comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in SEQ ID NO:2, in which the tyrosine at position 33 and the tyrosine at position 36 in SEQ ID NO:2 are sulfated, inhibits thrombin with an inhibition constant Ki of about 2 nM.

[0137] Determination of anticoagulant activity The anticoagulant activity (thrombin time, TT) of the peptides disclosed herein is determined by measuring their ability to slow the clotting of human plasma in vitro using a clinical TT assay. Briefly, human plasma (800 μL) from healthy donors is mixed with a range of peptide concentrations, clotting is initiated by the addition of thrombin, and clotting time is measured using a coagulometer. Peptides that extend clotting time to ≥ 30 seconds at a concentration of 50 nM can be further examined in vitro / ex vivo for activated partial thromboplastin time (aPTT). Briefly, pooled citrated plasma from C57BL6 / J mice is pre-incubated with various concentrations (0-12 μg / mL) of peptide. The aPTT of each plasma sample is quantified after the addition of coagulation activators and CaCl2. For the ex vivo assay, mice are injected iv with peptide (at a fixed concentration determined from in vitro aPTT) and whole blood is collected into sodium citrate (approximately 130 μL) at 0, 15, 30, 45, 60, 90 and / or 120 minutes after administration. aPTT is quantified on isolated plasma using a Siemans aPTT kit (Actin FS and CaCl2 solution), with fibrin production monitored to measure clotting time.

[0138] The anticoagulant activity (aPTT) of the peptides disclosed herein is determined by measuring their ability to slow the clotting of human plasma in vitro using a clinical aPTT assay (Siemens Healthineers, Erlangen, Germany). Human plasma (lyophilized powder from Stago, France) can be used to determine the dose response of the peptides. Human plasma (100 μL) is mixed with a range of peptides (0-1000 μg / ml). aPTT is quantified for the mixed plasma by a BFT II analyzer (Siemens) using turbodensitometric detection technology.

[0139] Determination of clotting time and blood loss In the Examples, the inventors show that the above described peptides have potent in vivo antithrombotic activity with reduced clotting times and reduced blood loss compared to known anticoagulants. This is advantageous since many antithrombotic drugs are limited in use due to the risk of bleeding, including intracranial bleeding in stroke therapy.

[0140] Blood loss is a clinically significant problem because the bleeding risk is significantly increased in patients undergoing anticoagulant therapy. In particular, there is no effective treatment available for bleeding caused by known anticoagulants, including hirudin. Thus, there is a need for agents that reduce blood loss.

[0141] The present inventors demonstrate the surprising effect that sulfated polypeptides according to the invention show significantly less bleeding compared to the known anticoagulant hirudin.

[0142] In one embodiment, the peptide of the present invention reduces clotting time by at least 20% based on molar equivalents of peptide and known thrombin inhibitor compared to known thrombin inhibitors that result in significant blood loss as measured using an in vitro activated partial thromboplastin time (aPTT) assay. Preferably, the clotting time is reduced by more than 20%, more than 30%, more than 40%, more than 50% or more than 60% compared to known thrombin inhibitors. Preferably, the known thrombin inhibitor is hirudin or argatroban, more preferably hirudin.

[0143] In one embodiment, the peptide of the invention comprises a clotting time of about 60 seconds to about 200 seconds at a concentration of greater than about 4 μg / mL, as measured using an in vitro activated partial thromboplastin time (aPTT) assay. Preferably, the clotting time is about 60 seconds to about 150 seconds, more preferably about 60 seconds to about 100 seconds.

[0144] In one embodiment, the peptides of the invention provide a clotting time of about 60 seconds to about 200 seconds when administered to an individual in an amount of about 1 μg / kg of the individual to about 10 mg / kg of the individual, as measured ex vivo using an in vitro activated partial thromboplastin time (aPTT) assay. Preferably, the clotting time is about 60 seconds to about 150 seconds, more preferably about 60 seconds to about 100 seconds. The amount of peptide can be at least about 1 μg / kg, 5 μg / kg, 10 μg / kg, 20 μg / kg, 50 μg / kg, 100 μg / kg, 250 μg / kg, 500 μg / kg, 750 μg / kg, 1 mg / kg, 2 mg / kg, 5 mg / kg, or 10 mg / kg.

[0145] In one embodiment, the peptides of the invention do not result in significant blood loss. Preferably, the peptides do not result in significant blood loss as measured using any of the assays described herein capable of determining blood loss. More preferably, the peptides, when administered to an individual, reduce blood loss by at least 10-fold based on molar equivalents of peptide and known thrombin inhibitor compared to known thrombin inhibitors that result in significant blood loss. In another embodiment, the peptides of the invention reduce blood loss by at least 20-fold, 50-fold, 100-fold, 1000-fold or more compared to known thrombin inhibitors. Preferably, the known thrombin inhibitor is hirudin or argatroban, more preferably hirudin. .

[0146] Treatment methods The disclosed peptides and compositions can be used to inhibit thrombin activity in antithrombotic amounts sufficient to inhibit thrombin activity in a subject, such as a human, in which pathological thrombosis is not desired. Thus, the peptides of the invention are useful for preventing or treating thromboembolic conditions in mammals.

[0147] Accordingly, there is provided a method of preventing or treating a thromboembolic condition in an individual in need thereof, comprising, consisting essentially of, or consisting of administering to the individual a therapeutically effective amount of a peptide of the invention, thereby preventing or treating the thromboembolic condition in the individual.

[0148] The present invention also provides a method of treating a thromboembolic condition or reducing the severity of symptoms of a thromboembolic condition in an individual, comprising, consisting essentially of, or consisting of administering to an individual in need thereof a therapeutically effective amount of a peptide of the invention, thereby treating the thromboembolic condition or reducing the severity of symptoms of a thromboembolic condition in the individual.

[0149] In another aspect, the present invention also provides a method for inhibiting or minimizing the progression of symptoms of a thromboembolic condition in an individual, comprising, consisting essentially of, or consisting of administering a therapeutically effective amount of a peptide of the invention to an individual experiencing symptoms of a thromboembolic condition, thereby inhibiting or minimizing the progression of symptoms of the thromboembolic condition in the individual.

[0150] Thus, in a further aspect, the present invention provides a method of reducing the severity of a thromboembolic condition in an individual in need thereof, comprising, consisting essentially of or consisting of the step of administering to the individual in need thereof a therapeutically effective amount of a peptide of the invention, thereby reducing the severity of the thromboembolic condition in the individual.

[0151] The term "thromboembolic conditions" as used herein includes myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, disseminated intravascular coagulation, cardiovascular and cerebrovascular thrombosis, thrombosis associated with postoperative trauma, obesity, pregnancy, side effects of oral contraceptives, hypercoagulable states associated with prolonged immobilization and hematological, immune or rheumatic disorders, unstable angina, arteriosclerosis, reocclusion of blood vessels after angioplasty with a balloon catheter or blood clotting in hemodialysis, artificial heart valves, extracorporeal membrane oxygenation or stent placement.

[0152] Symptoms of thromboembolic conditions may be determined by symptoms and will be clear to those skilled in the art. Examples of stroke symptoms include: weakness or numbness or paralysis of the face, arms or legs, on one or both sides of the body; speech disorder; dizziness; balance disorder; visual disorder; headache; acute delirium; cognitive impairment. Preferably, the individual is identified as having or suspected of having one or more symptoms of a thromboembolic condition, preferably at least two, three or four symptoms of a thromboembolic condition.

[0153] In addition to its effects on the coagulation process, thrombin has been shown to activate many cells, including neutrophils, fibroblasts, endothelial cells, smooth muscle cells, etc. Thus, the peptides of the invention may also be useful in the treatment or prevention of adult respiratory distress syndrome, septic shock, sepsis, inflammatory responses (including but not limited to edema), acute or chronic atherosclerosis, and reperfusion injury.

[0154] In another aspect, the present invention also provides a method for inhibiting or minimizing the progression of blood clot formation associated with inflammation caused by viral infection in an individual, comprising, consisting essentially of, or consisting of administering a therapeutically effective amount of a peptide of the present invention to an individual experiencing inflammation caused by viral infection, thereby inhibiting or minimizing the progression of blood clot formation in the individual.

[0155] Thus, in a further aspect, the present invention provides a method of reducing the severity of blood clot formation associated with inflammation caused by viral infection in an individual in need thereof, comprising, consisting essentially of or consisting of the step of administering to the individual in need thereof a therapeutically effective amount of a peptide of the present invention, thereby reducing the severity of blood clot formation in the individual.

[0156] The virus is preferably selected from coronavirus, influenza, parainfluenza, respiratory syncytial virus (RSV), adenovirus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV), dengue virus, rhinovirus, herpes simplex virus and enterovirus. More preferably, the virus is coronavirus or influenza. Even more preferably, the virus is severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), most preferably SARS-CoV-2.

[0157] The peptides of the invention may also be used as anticoagulants in extracorporeal blood circuits such as those required in dialysis and surgery (such as coronary artery bypass surgery).

[0158] The peptides of the present invention can be used as monotherapy. In another embodiment, the peptides of the present invention can be administered in combination with one or more thrombolytic agents, one or more platelet inhibitors, or a combination thereof. The present invention provides a dose of thrombolytic agent and / or platelet inhibitor when used in combination with the peptide at or below the dose prescribed according to the approved indication.

[0159] As described above, at least 20-30% of patients undergoing rtPA therapy for ischemic stroke will experience complete arterial recanalization following rtPA therapy, and 20-30% of these will experience reocclusion. Some believe this occurs when rtPA therapy exposes clot-trapped thrombin (which is known to bind to clots via exosite II), allowing the active site of thrombin to cleave fibrinogen and fibrinopeptides to amplify and build the clot. Thus, thrombin inhibitors as described herein can be provided to individuals undergoing rtPA therapy to allow elution of clot-bound thrombin from fibrin clots, thereby minimizing the amount of thrombin in the clot that would otherwise cause fibrin generation and clot expansion.

[0160] Thus, in one embodiment, the peptides of the invention can be used in combination with thrombolytic agents, such as tissue plasminogen activator (natural or recombinant), streptokinase, urokinase, pro-urokinase, anisolated streptokinase-plasminogen activator complex (ASPAC), animal salivary plasminogen activator, and the like. The peptides of the invention can act in a synergistic manner to prevent reocclusion and / or reduce the time to re-flow after successful thrombolytic therapy. The peptides of the invention can also allow a reduction in the dose of the thrombolytic agent used, thus minimizing the possibility of hemorrhagic side effects. In a preferred embodiment, the thrombolytic agent is rtPA.

[0161] The peptides of the present invention may also be used in combination with platelet inhibitors such as aspirin; triflusal; adenosine diphosphate receptor inhibitors including cangrelor, clopidogrel, prasugrel, ticagrelor, ticlopidine; phosphodiesterase inhibitors such as cilostazol; protease-activated receptor-1 antagonists such as vorapaxar; glycoprotein IIB / IIA inhibitors such as abciximab, eptifibatide, tirofiban; adenosine reuptake inhibitors such as dipyridamole; and thromboxane inhibitors.

[0162] The peptides of the invention can be used in combination with thrombolytic agents, preferably tissue plasminogen activator and platelet inhibitors.

[0163] composition The pharmaceutical composition can be formulated for any suitable route of administration, preferably parenteral administration.The term "parenteral" as used herein includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection and any similar injection or infusion technique.Typically, the thrombin inhibitor peptide is provided in the form of a composition adapted for iv administration.

[0164] Methods for preparing peptides into suitable forms (e.g., pharmaceutical compositions) for administration to a subject are known in the art and include, for example, those methods described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and the US Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0165] The pharmaceutical compositions of the present invention are particularly useful for parenteral administration, e.g., intravenous administration or administration into a cavity or lumen of an organ or joint. Compositions for administration generally comprise a solution of the peptide of the present invention dissolved in a pharma- ceutically acceptable carrier, e.g., an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline, and the like. The compositions can contain pharma- cetically acceptable auxiliary substances, as necessary, to approximate physiological conditions, e.g., pH adjusting and buffering agents, toxicity adjusting agents, and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the peptide of the present invention in these formulations can vary over a wide range and is selected primarily based on fluid volumes, viscosities, body weight, and the like, according to the particular mode of administration selected and the needs of the patient. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous excipients, such as oil mixtures and ethyl oleate, can also be used. Liposomes can also be used as carriers. The excipients can contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

[0166] In one embodiment, the peptide is administered locally to the site where the thrombus is forming.

[0167] In another embodiment, the peptide is administered directly to the thrombus.

[0168] In another embodiment, the peptide is administered as a bolus. The bolus can be an intravenous bolus or a bolus + infusion to maintain a steady state level. Preferably, the peptide is administered to an individual according to the invention within 12 hours, preferably within 3 hours, 2 hours, 1 hour or less, after the first identification of a thromboembolic condition.

[0169] Administration is preferably by bolus injection or intravenous infusion, preferably as soon as possible after confirmation of thromboembolism.

[0170] The amount administered will depend on the age, health and weight of the individual.

[0171] Typically, the thrombin inhibitor peptide is provided in an amount of about 1 μg / kg to 10 mg / kg of recipient. EXAMPLES

[0172] Example 1 – Peptide synthesis The peptides of SEQ ID NO:1 and SEQ ID NO:2 can be prepared by chemical synthesis, including diselenide selenoester linkage and deselenation, and by solid phase peptide synthesis, as described herein.

[0173] material Peptide grade dimethylformamide (DMF) was obtained from Labscan. Amino acids, coupling reagents and resins for Fmoc-solid phase peptide synthesis (SPPS) were obtained from Novabiochem or GL Biochem. SPPS was performed in polypropylene syringes equipped with Teflon filters purchased from Torviq. Analytical reversed-phase high performance liquid chromatography (HPLC) was performed on a Waters Acquity UPLC system equipped with a PDA eλ detector (λ = 210-400 nm), Sample Manager FAN and Quaternary Solvvent Manager (H-class) module or a Waters System 2695 Separation Module equipped with a 2996 Photodiode Array Detector. Peptides were analyzed at 0.2 mL min on the HPLC system. -1 The XBridge BEH 5 μm, 2.1 × 150 mm wide pore column (C-18) was run at a flow rate of 0.6 mL min on a UPLC system. -1Analyses were performed using a Waters Acquity UPLC BEH 1.7 μm 2.1×50 mm column (C-18) at a flow rate of 100 Hz. Both instruments were run using a mobile phase consisting of a linear gradient of 0.1% trifluoroacetic acid in HO (solvent A) and 0.1% trifluoroacetic acid in acetonitrile (solvent B) as indicated. Analysis of chromatograms was performed using Empower 3 Pro software (2010) and retention times (Rt min) of pure peptides and proteins are reported using the gradients specified.

[0174] Preparative and semi-preparative reversed-phase HPLC were performed using a Waters 600E Multisolvent Delivery System equipped with a Rheodyne 7725i injection valve (4 mL loading loop) and a Waters 500 pump, with a Waters 490E programmable wavelength detector operating at 214, 230, 254 or 280 nm. Preparative reversed-phase HPLC was performed at a flow rate of 7 mL min -1 At a flow rate of Waters The semi-preparative reversed-phase HPLC was performed using a Sunfire C18 column (5 μm, 10 × 250 mm). -1 The purification was carried out using a Waters XBridge-BEH300 wide-pore C18 column (5 μm, 10 × 250 mm) at a flow rate of 100 s. Peptides and proteins with free sulfated tyrosine (sTyr) residues were purified using a mobile phase of 0.1% formic acid (in water) (solvent A) and 0.1% formic acid (in acetonitrile) (solvent B) using the indicated linear gradient. In all other cases using the specified linear gradient, a mobile phase of 0.1% trifluoroacetic acid (in water) (solvent A) and 0.1% trifluoroacetic acid (in acetonitrile) (solvent B) was used. After lyophilization, peptides were isolated as TFA or formate salts depending on the chromatographic eluent.

[0175] The LC-MS was performed using an LC-M20A pump and an SPD-20A coupled to a Shimadzu 2020 mass spectrometer (ESI) operated in positive mode unless otherwise stated. On a Shimadzu LC-MS 2020 instrument consisting of a UV / Vis detector or SP Separation was performed on a Shimadzu UPLC-MS equipped with the same modules as the LC-MS system except for a D-M30A diode array detector. Separation was performed on the LC-MS system at a flow rate of 0.2 mL min -1 Separation on the UPLC-MS system was performed on a Waters Sunfire 5 μm, 2.1 × 150 mm column (C-18) or wide-pore equivalent, operated at a flow rate of 0.6 mL min -1 At a flow rate of Waters An Acquity UPLC BEH 1.7 μm 2.1 × 50 mm column (C-8) was used for separation. Separation was performed using mobile phases of 0.1% formic acid (in water) (solvent A) and 0.1% formic acid (in acetonitrile) (solvent B) with a linear gradient of 0-50% B over 30 min on the LC-MS system and 0-50% B over 8 min on the UPLC-MS system.

[0176] Low-resolution mass spectra were recorded on a Shimadzu 2020 mass spectrometer (ESI) operated in positive or negative mode as indicated. Low-resolution MALDI-TOF mass spectra were recorded on a Bruker Autoflex™ Speed ​​MALDI-TOF mass spectrometer operated in linear mode using a matrix of 10 mg / mL sinapinic acid (1:1 v / v in water / acetonitrile) without TFA.

[0177] General Peptide Synthesis Procedure General procedure for Fmoc strategy SPPS (100-200 μmol scale) 2-Chlorotrityl chloride resin loading 2-Chlorotrityl chloride resin (1.22 mmol / g loading) was swollen in dry CHCl for 30 min followed by washing with CHCl (10×3 mL). A solution of Fmoc-Xaa-OH (0.7 mmol / g resin) and iPrNEt (4 equivalents relative to resin functionalization) in CHCl (final concentration of amino acids 0.125 M) was added to the resin, which was shaken at room temperature for 16 h. The resin was then washed with CHCl (5×3 mL), DMF (5×3 mL) and CHCl (5×3 mL). The resin was then capped via treatment with 17:2:1 v / v / v CHCl:MeOH:iPrNEt (5 mL) for 40 min at room temperature. The resin was then washed again with CH2Cl2 (5 x 3 mL), DMF (5 x 3 mL) and CH2Cl2 (5 x 3 mL) before determination of the putative loading of the first amino acid.

[0178] Rink Amide Resin Loading Rink amide resin (0.7 mmol / g loading) was swollen in dry CHCl for 30 min followed by washing with CHCl (10×3 mL). The resin was treated with piperidine / DMF (1:4 v / v, 3 mL, 2×5 min) and then washed with DMF (5×3 mL), CHCl (5×3 mL) and DMF (5×3 mL). A solution of Fmoc-Xaa-OH (0.7 mmol / g resin), PyBOP (4 equivalents relative to resin functionalization) and N-methyl-morpholine (8 equivalents relative to resin functionalization) in DMF or NMP (final concentration of amino acids 0.125 M) was added to the resin, which was shaken for 2 h at room temperature. The resin was then washed with DMF (5×3 mL) and CHCl (5×3 mL) before capping via treatment with 9:1 v / v pyridine:AcO (5 mL) for 5 min at room temperature. The resin was then washed again with CHCl (5×3 mL), DMF (5×3 mL) and CHCl (5×3 mL) before determination of the putative loading of the first amino acid.

[0179] Loading estimate of the first amino acid The resin was treated with piperidine / DMF (1:4 v / v, 3 mL, 2 x 5 min) and then washed with DMF (5 x 3 mL), CH2Cl2 (5 x 3 mL) and DMF (5 x 3 mL). The combined deprotection solutions were then diluted 1:1 with fresh piperidine / DMF (1:4 v / v). The solution was diluted 50-100 times with fresh piperidine / DMF (1:4 v / v) and the UV absorbance of the piperidine-fulvene adduct was measured (λ = 301 nm, ε = 7800 M -1 cm -1 ), to estimate the amount of amino acid loaded onto the resin.

[0180] Peptide assembly via iterative SPPS Peptides were assembled by stepwise Fmoc-SPPS on a 0.05-0.2 mmol scale. Coupling of each amino acid (4 eq.) was achieved using PyBOP (4 eq.) and NMM (8 eq.) in DMF (0.1 M) for 1 h at room temperature. Unless otherwise specified, after all steps a capping step was introduced using 0.3 M acetic anhydride and 0.3 M iPr2NEt in DMF for 3 min at room temperature. Fmoc-deprotection steps were performed by treatment with 20% v / v piperidine in DMF (2 × 5 min) at room temperature. After each coupling, capping or deprotection step the resin was washed with DMF (5 × 3 mL), CHCl2 (5 × 3 mL) and DMF (5 × 3 mL).

[0181] Coupling of Fmoc-Tyr[SO3-CH2C(CH3)3]-OH A solution of amino acid (2.0 equiv.), DIC (2.0 equiv.) and HOAt (2.0 equiv.) in DMF (0.1 M) was added to the resin and shaken at room temperature for 16 h. The resin was then washed with DMF (5×3 mL), CHCl (5×3 mL) and DMF (5×3 mL) and the capping step was carried out as described above. Iterative Fmoc-SPPS was then used to complete the synthesis of the desired fragment.

[0182] Coupling of Boc-Asp(SePMB)-OH or Boc-Sec(PMB)-OH A solution of amino acid (1.5 equiv.), DIC (1.5 equiv.) and HOAt (1.5 equiv.) in DMF (0.05 M) was added to the resin and shaken at room temperature for 16 h. The resin was then washed with DMF (5×3 mL), CH2Cl2 (5×3 mL) and DMF (5×3 mL).

[0183] Peptide assembly via iterative fully automated microwave-assisted SPPS Peptides were assembled by stepwise microwave-assisted Fmoc-SPPS on a Protein Technologies Symphony peptide synthesizer operating on a 0.1-0.2 mmol scale. Activation of the Fmoc-protected amino acids (0.3 M solution in DMF) was performed using 0.3 M Oxyma in DMF / 0.3 M DIC in DMF (1:1:1 molar ratio) in 4 equivalent excess over the initial resin loading. Coupling steps were performed at 25 °C for 45 min. Unless otherwise specified, a capping step was introduced after each coupling step and was performed by treatment with 0.3 M acetic anhydride / 0.3 M iPr2NEt solution in DMF (1 × 3 min). Fmoc-deprotection steps were performed by treatment with 20% v / v piperidine in DMF (2 × 3 min) at room temperature. After each coupling, capping or deprotection step, the resin was washed with DMF (4 × 30 s). Once the peptide chain was fully assembled, the resin was washed with CH2Cl2 (5 x 30 s) and gently dried under a stream of nitrogen.

[0184] Cutting from resin Fully deprotected peptide The resin-bound peptide was dissolved in an ice-cold mixture of TFA:iPrSiH:water (90:5:5 v / v / v, 5 mL) and shaken at room temperature for 2 h. At this point the resin was filtered and washed with fresh cleavage cocktail. The combined cleavage solutions were worked up as described below.

[0185] Side-chain protected peptides The resin was washed with CHCl (5×3 mL) and then treated with a solution of HFIP / CHCl (3:7 v / v, 4 mL) and shaken at room temperature for 40 min. The resin was filtered and washed with CHCl (3×3 mL). The combined cleavage solution and washings were concentrated under a stream of nitrogen to give the crude side-chain protected product.

[0186] Liquid-phase selenoesterification A solution of the fully side-chain protected peptide in dry DMF (20 mM) was treated with diphenyl diselenide (10 equiv.) and tributylphosphine (10 equiv.) under an argon atmosphere for 4-5 h at −30° C. The solution was then concentrated under a stream of nitrogen and subjected to the acidic deprotection conditions described for the fully deprotected peptide.

[0187] Post-treatment and purification Ice-cold diethyl ether (30 mL) was added dropwise to the concentrated cleavage solution to precipitate the crude peptide. The precipitate was then collected via centrifugation and washed with additional diethyl ether to remove any remaining scavengers. Residual diethyl ether was removed under a gentle stream of nitrogen and dissolved in 0.1% v / v TFA aqueous buffer (minimal addition of MeCN was made to aid dissolution when necessary). The crude peptide was analyzed by LC-MS (ESI) and purified by RP-HPLC.

[0188] On-resin Boc protection, allyl deprotection and selenoesterification After the synthesis of the sequence was completed, Fmoc deprotection was carried out as described above. The resin (10-90 μmol) was then treated with Boc anhydride (2 equiv., 4-40 mg) and pyridine (2 equiv., 2-14.5 μL) in DMF (0.01-0.03 M) for 4 h at room temperature. The resin was then washed as described above and treated with Pd(PPh3)4 (0.9 equiv., 10-100 mg) and phenylsilane (40 equiv., 50-450 μL) in DCM (0.01-0.03 M) for 1 h at room temperature. This procedure was then repeated once and the resin was washed as described above. Finally, the resin was treated with DPDS (30 equiv., 90-850 mg) and Bu3P (30 equiv., 75-675 μL) in DMF (0.01-0.03 M) for 3 h at 0° C. The resin was washed again as described above before being subjected to the overall cleavage conditions.

[0189] PMB deprotection and purification of Ct fragment The crude C-terminal fragment was dissolved (7 mg / mL) in a 12:5:3 v / v / v mixture of TFA:buffer (6 M Gn.HCl, 100 mM HEPES, pH 4-5):DMSO and allowed to stir at room temperature for 1-4 h, then diluted with water before purification by RP-HPLC.

[0190] General protocol for one-pot coupling-deselenation A 5 mM solution of peptide diselenide dimer (1.0 equiv.) in ligation buffer (6 M Gn.HCl, 100 mM Na2HPO4, adjusted to pH 6.3) was added to the appropriate peptide selenoester (2.0-3.0 equiv.). The resulting solution was carefully readjusted to pH 6.2-7.5 with 1 M KOH, and the reaction was incubated at 25°C for 20 min. Upon complete conversion to the corresponding ligation product (as determined by UPLC-MS monitoring), a solution of TCEP (500 mM) and dithiothreitol (50 mM) in buffer (6 M Gn.HCl, 100 mM Na2HPO4, pH 4-5) was added to give a final concentration of 250 mM TCEP, 25 mM dithiothreitol, and 2.5 mM for the peptide diselenide dimer starting material. The reaction was then incubated at 25°C for 10 min. After UPLC-MS analysis showed complete conversion to the deselenium product, the reaction mixture was incubated at 37° C. for 6 h to remove the neopentyl group. The ligation mixture was then diluted and analyzed by RP-HPLC. Purified.

[0191] Example 1A: Synthesis of SEQ ID NO:1 via diselenide-selenoester ligation (DSL) and deselenation Synthesis of SEQ ID NO:1 (Madanin-like 2) fragment [ka] MadL2(1-30) selenoesters were prepared following the Fmoc strategy SPPS by a combination of automated and manual synthesis on 2-chlorotrityl chloride resin (100 μmol) with the N-terminal amino acid coupled as the Boc-protected amino acid. Upon completion of the resin-bound peptide fragment, HFIP-mediated cleavage and solution-phase selenoesterification with diphenyl diselenide (DPDS) (927 mg, 30 equiv.) were completed as outlined in the general procedure. Removal of all acid-labile protecting groups was achieved via treatment of the crude peptide selenoester in a solution of TFA / iPr3SiH / HO (90:5:5 v / v / v, 4 mL) for 2 h at room temperature. The deprotected solution was then concentrated under a stream of nitrogen and the crude selenoester was precipitated from ice-cold diethyl ether. Purification of the crude peptide by preparative RP-HPLC (0–30% B, 0.1% v / v TFA over 40 min) afforded the MadL2(1–30) selenoester as a fluffy white solid after lyophilization (27.88 mg, 7.98% yield).

[0192] [ka] MadL2 DS(31-60) diselenide was prepared on 2-chlorotrityl chloride resin (50 μmol) following the Fmoc strategy SPPS by a combination of automated and manual synthesis. Removal of all acid-labile protecting groups was achieved via treatment of the crude peptide in a solution of TFA / iPr3SiH / H2O (90:5:5 v / v / v, 4 mL) for 2 h at room temperature. This deprotected solution was then concentrated under a stream of nitrogen and the crude peptide was precipitated from ice-cold diethyl ether. The PMB group was then removed as described above. Preparative RP-HPLC (0-50% B, 0.1% v / v TFA over 40 min) was used to determine the cleavage site. ) Purification of the crude peptide afforded the MadL2 DS(31–60) diselenide as a fluffy white solid after lyophilization (4.36 mg, 2.24% yield).

[0193] SEQ ID NO:1 (MadL2)DS Synthesis scheme of SEQ ID NO:1 (MadL2)DS via DSL-deselenation [ka] One-pot peptide ligation of MadL2(1–30) selenoester (2.7 mg, 0.77 μmol) and MadL2 DS(31–60) diselenide (2.0 mg, 0.52 μmol) followed by in situ deselenation was carried out by replacing 10% of the buffer volume with DMF. Purification via preparative HPLC (0-40% B over 40 min, 0.1% v / v formic acid) followed by lyophilization afforded the final protein MadL2 DS as a white solid (1.99 mg, 55%). Analytical HPLC (purified final product): Rt 3.84 min (0-50% B over 5 min, 0.1% v / v TFA, λ=214 nm); mass calculated [M+4H] 4+ :1747.5[M+5H] 5+ :1398.2[M+6H] 6+ :1165.4, [M+7H] 7+ :999.0, [M+8H] 8+ :874.3, [M+9H] 9+ :777.2 Mass actual value (ESI) 1748.9[M+4H] 4+ , 1399.2[M+5H] 5+ , 1166.2[M+6H] 6+ , 999.8[M+7H] 7+ , 874.9[M+8H] 8+ , 777.9[M+9H] 9+ . High resolution (ESI+):C 285 H 437 N 85 O 117 Calculated value for S2: [M+8H] 8+ , 874.89037. Measured value: 874.88897[M+8H] 8+ . Difference: 1.6ppm.

[0194] Example 1B: Synthesis of SEQ ID NO:2 via diselenide-selenoester ligation (DSL) and deselenation Synthesis of SEQ ID NO:2 (Madanin-like 1) fragment [ka] MadL1(1-28) selenoester was prepared by loading of Fmoc-Val-OH onto 2-chlorotrityl chloride resin (62.5 μmol) and extended via Fmoc strategy SPPS by a combination of automated and manual synthesis, with the N-terminal amino acid coupled as the Boc-protected amino acid. Upon completion of the resin-bound peptide fragment, HFIP-mediated cleavage and solution-phase selenoesterification with diphenyl diselenide (DPDS) (590 mg, 30 equiv.) were completed as outlined in the general procedure. Removal of all acid-labile protecting groups was achieved via treatment of the crude peptide selenoester in a solution of TFA / iPr3SiH / HO (90:5:5 v / v / v, 4 mL) for 2 h at room temperature. The deprotected solution was then concentrated under a stream of nitrogen and the crude selenoester was precipitated from ice-cold diethyl ether. Purification of the crude peptide by preparative RP-HPLC (0% B for 5 min, 0%–15% B over 5 min, then 15%–35% B over 40 min, 0.1% v / v TFA) afforded the MadL1(1–28) selenoester as a fluffy white solid after lyophilization (56 mg, 27% yield).

[0195] [ka] MadL1 DS(29-61) diselenide was prepared on 2-chlorotrityl chloride resin (50 μmol) following the Fmoc strategy SPPS by a combination of automated and manual synthesis. Removal of all acid-labile protecting groups was achieved via treatment of the crude peptide in a solution of TFA / iPr3SiH / H2O (90:5:5 v / v / v, 4 mL) for 2 h at room temperature. The deprotected solution was then concentrated under a stream of nitrogen and the crude peptide was precipitated from ice-cold diethyl ether. The PMB group was then removed as described above. Purification of the crude peptide by preparative RP-HPLC (0%–35% B over 5 min, 35% B over 5 min, followed by 35%–55% B over 40 min, 0.1% v / v TFA) afforded the MadL1 DS(29–61) diselenide as a fluffy white solid after lyophilization (6.2 mg, 3% yield).

[0196] SEQ ID NO:2 (MadL1)DS Synthesis scheme of SEQ ID NO:2 (MadL1)DS via DSL-deselenation [ka] One-pot peptide ligation of MadL1 (1–28) selenoester (2.5 mg, 0.75 μmol) and MadL1 DS (29–61) diselenide (2.0 mg, 0.50 μmol) followed by in situ deselenation was carried out according to the general procedure. Purification via preparative HPLC (0% for 5 min, 0–40% B over 40 min, 0.1% v / v formic acid) followed by lyophilization afforded the final protein MadL1 DS as a white solid (2.1 mg, 67%). Analytical HPLC (purified final product): Rt 26.0 min (0–50% B over 30 min, 0.1% v / v TFA, λ=214 nm); mass calculated [M+4H] 4+ :1740.0[M+5H] 5+ :1392.2[M+6H] 6+ :1160.4 Mass actual value (ESI) 1740.3[M+4H] 4+ , 1392.4[M+5H] 5+ , 1160.5[M+6H]6+ .

[0197] Example 1C: Synthesis of SEQ ID NO:1 via solid phase peptide synthesis Synthesis of SEQ ID NO:1 (MDL2)(1-60)Y32,Y35 on a single bead via SPPS [ka] MadL2(1-60) Y32, Y35 was synthesized by cleaving the Y32 and Y35 positions ( [ka] Neopentyl-protected tyrosine sulfate was incorporated into the 100 μmol of Fmoc-Ala-Wang polystyrene resin via Fmoc-SPPS at 50° C. on a SYRO peptide synthesizer using the protocol described below. To prevent aspartimide formation, the Fmoc-Asp(OtBu)-(Dmb)Gly-OH dipeptide was also incorporated as needed using the same synthesis conditions ( [ka] (denoted as ). Removal of all acid-labile protecting groups with simultaneous cleavage from the resin support was achieved via treatment in a solution of TFA / iPr3SiH / HO (90:5:5 v / v / v, 4 mL) for 2 h at room temperature. The deprotected solution was then concentrated under a stream of nitrogen and the crude peptide was precipitated from ice-cold diethyl ether. Purification of this crude peptide by preparative RP-HPLC (0–40% B, 0.1% v / v TFA over 40 min) afforded MadL2(1–60) bearing neopentyl-protected sTyr residues at Y32, Y35 as a fluffy white solid after lyophilization. The neopentyl-protected peptide was then dissolved in 6 M Gn.HCl, 0.1 M Na2HPO4, 250 mM TCEP, 25 mM DTT (pH 5) to a concentration of 2.5 mM and incubated at 37 °C for 16 h to remove the neopentyl protecting group, followed by preparative RP-HPLC (0-40% B over 40 min, 0.1% v / v formic acid) to yield MadL2(1-60)Y32,Y35 as fluffy white solids after lyophilization. Yield: 40.6 mg, 5.7%. NOTE: All characterization data were consistent with that obtained for material derived from the coupling strategy.

[0198] Biotage SYRO I Automated Peptide Synthesizer Protocol Fmoc deprotection: The resin was treated with piperidine:DMF (2 x 4 min, 1.6 mL of 20%). The resin was filtered and washed with DMF (4 x 1.7 mL).

[0199] Coupling of proteinogenic amino acids: Fmoc-AA-OH (0.8 mL, 0.5 M), DIC (0.8 mL, 0.5 M) and Oxyma (0.8 mL, 0.55 M) were dispensed from stock solutions (in DMF) onto the resin. The reaction was heated to 50° C. and mixed for 30 min. The resin was filtered and washed with DMF (4×2.5 mL).

[0200] Capping: The resin was treated with a capping solution containing 5% acetic anhydride and 10% iPr2NEt in DMF (0.8 mL) for 6 min. The resin was filtered and washed with DMF (4 x 2.5 mL).

[0201] Example 2 – Biological Data Inhibition of thrombin by sulfated forms of SEQ ID NO:1 and SEQ ID NO:2 Inhibition of the amidolytic activity of human α-thrombin (Haematologic Technologies) was followed spectrophotometrically using Tos-Gly-Pro-Arg-p-nitroanilide (Chromozym TH; Roche) as a chromogenic substrate. Inhibition assays were performed using 0.2 nM enzyme, 100 μM substrate, and increasing concentrations of peptide. The concentration of each peptide was determined using a Direct Detect infrared spectrometer (Millipore). The inhibition constant (Ki) of each peptide was determined according to the tight binding model by fitting the steady-state kinetic inhibition data to the Morrison equation (Williams JW & Morrison JF (1979)). All reactions were carried out in 96-well microtiter plates in 50 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mg / mL BSA at 37°C. Reaction progress was monitored at 405 nm for 1-2 h on a Synergy2 Multimode microplate reader (BioTek). Dose-response curves were used to determine Ki values ​​using Prism 8.0 (GraphPad Software). At least two independent experiments with duplicate reactions were performed for each peptide along with control reactions in the absence of enzyme. The goodness-of-fit parameters R2 for all curves were between 0.989 and 0.999.

[0202] The inventors have demonstrated that the sulfated peptide of SEQ ID NO:1 has a K of less than about 3 nM, as shown in FIG. i Furthermore, a doubly sulfated variant of this peptide is more than two orders of magnitude more potent as an inhibitor of human thrombin in vitro than its non-sulfated counterpart.

[0203] The inventors have demonstrated that the sulfated peptide of SEQ ID NO:2 has a K of less than about 20 nM, as shown in FIG. i Furthermore, a doubly sulfated variant of this peptide is an order of magnitude more potent as an inhibitor of human thrombin in vitro than its non-sulfated counterpart.

[0204] Clotting and bleeding times of peptide of SEQ ID NO:1 Mouse plasma activated partial thromboplastin time (aPPT) dose response curves for SEQ ID NO:1 were compared with hirudin and argatroban.

[0205] The anticoagulant activity (thrombin time, TT) of the peptides disclosed herein is determined by measuring their ability to slow the clotting of human plasma in vitro using a clinical TT assay. Briefly, human plasma (800 μL) from healthy donors is mixed with a range of peptide concentrations, clotting is initiated by the addition of thrombin, and clotting time is measured using a coagulometer. Peptides that extend clotting time to ≥ 30 seconds at a concentration of 50 nM can be further examined in vitro / ex vivo for activated partial thromboplastin time (aPTT). Briefly, pooled citrated plasma from C57BL6 / J mice is pre-incubated with various concentrations (0-12 μg / mL) of peptide. The aPTT of each plasma sample is quantified after the addition of coagulation activators and CaCl2. For the ex vivo assay, mice are injected iv with peptide (at a fixed concentration determined from in vitro aPTT) and whole blood is collected into sodium citrate (approximately 130 μL) at 0, 15, 30, 45, 60, 90 and / or 120 minutes after administration. aPTT is quantified on isolated plasma using a Siemans aPTT kit (Actin FS and CaCl2 solution), with fibrin production monitored to measure clotting time.

[0206] The anticoagulant activity (aPTT) of the peptides disclosed herein was determined using a clinical aPTT assay ( The aPTT can be determined by measuring its ability to slow the clotting of human plasma in vitro using a lyophilized powder from Siemens Healthineers, Erlangen, Germany. Human plasma (lyophilized powder from Stago, France) can be used to determine the dose response of the peptide. Human plasma (100 μL) is mixed with a range of peptides (0-1000 μg / ml). The aPTT is quantified for the mixed plasma by a BFT II analyzer (Siemens) using a fast acting concentration detection technology.

[0207] We investigated the ability of peptide SEQ ID NO: 1 to prolong clotting time in vitro in an activated partial thromboplastin time (APTT) assay (Figure 3A). These studies demonstrate that while hirudin and argatroban exhibit AAPTs of greater than 200 seconds at concentrations above about 12 μg / mL, SEQ ID NO: 1 remained within a safer clotting time range with an AAPT of about 60 to about 200 seconds at concentrations of about 4 μg / mL to about 600 μg / mL.

[0208] SEQ ID NO:1 (MDL-2) clotting activity in vivo over time Mice were administered MDL-2 (10 mg / kg) and blood was collected via submandibular (cheek) bleeding over the indicated time course. For each sample, plasma was isolated and aPTT was quantified in vitro using the activated partial thromboplastin time (aPTT) assay as described herein.

[0209] plasma stability The plasma stability of the peptide of SEQ ID NO:1 (MDL-2) was determined using a modified method previously described by Teufel et al. (Teufel, Bennett et al. 2018). Positive control: propantheline bromide (10 mM stock in DMSO).

[0210] MDL-2 (5 mM stock (in 0.05 M aqueous NaOH)) and propantheline bromide were added to human plasma (citrated pooled plasma from Sigma Aldrich) to a final concentration of 200 μM. MDL-2 and positive controls were incubated at 37° C. for 0, 30, 60, 120 or 180 min and then quenched with three volumes of 1:1 v / v MeOH:MeCN. Samples were centrifuged at 13,500 rpm for 5 min after which aliquots of the supernatant (50 μL) were removed and analyzed by reversed-phase UHPLC and mass spectrometry. The area under the onset peak (normalized to the total area under the chromatogram) was used to quantitate the amount of remaining uncleaved peptide.

[0211] As shown in FIG. 3C, the peptide of SEQ ID NO:1 (MDL-2) remains stable in plasma for up to about 180 minutes.

[0212] blood loss Blood loss was assessed by performing a 3 mm tail cut (lop) in rodents to assess blood loss after compound / drug administration. Tail cuts were performed using a standard cut 3 mm from the tip of the tail (Schoenwaelder et al, 2011, Blood). The tail was immediately immersed in pre-warmed sterile saline (37°C) and observed for time to cessation of bleeding and rebleeding events for 30 min after cut. Blood samples were centrifuged at 4,000 rpm for 15 min and the pellet was resuspended in 2 ml of RBC lysis buffer for at least 30 min at room temperature. Blood loss was quantified using a hemoglobin assay kit as per the manufacturer's instructions (catalog no. MAK115; Sigma-Aldrich) using hemoglobin standards and measurements obtained using a CLARIOstar plate reader.

[0213] As shown in FIG. 4, peptide of sequence number 1 (MDL-2) at concentrations of 10 mg / kg or 20 mg / kg results in significantly reduced blood loss compared to 1 mg / kg hirudin (greater than about 2000 mg / dL) and 10 mg / kg argatroban (greater than about 2000 mg / dL).

[0214] Experimental thrombosis and thrombolysis models in rodents Thrombosis: Electrolytic injury was used to produce thrombotic occlusion of the carotid artery in situ in anesthetized mice as described (Jackson et al, Nature Medicine 2005; Schoenwaelder et al, J Biol Chem, 2007). Baseline blood flow through the carotid artery was measured using an ultrasound Doppler flow probe. The flow probe was removed and vascular injury was inflicted in the presence of hemostasis via placement of a distal hemostat (Micro Serrefine Clamp, 18055-05, Fine Science Tools, Canada). Delivery of electrical current (8 mA or 28 mA, 3 min) was achieved using a lesion creation device (Model 53500, Ugo Basile, Comerio, VA, Italy) while constantly flushing the injury site with saline to prevent tissue dehydration. After injury, the hemostat and electrodes were removed and the flow probe was reattached to the carotid artery to allow monitoring of blood flow for the remainder of the experimental procedure (60 min post-injury). Stable occlusion was defined as a reduction in blood flow to 0 ml / min / 100 g for 10 min.

[0215] Thrombolysis: Intravenous thrombolysis was initiated 15 minutes after stable thrombotic vessel occlusion as observed by flow probe. rtPA was administered (rtPA, 10 mg / kg bolus / infusion) via the jugular catheter using a Harvard Apparatus syringe pump (cat. no. 704504; Pump Elite 11 I / W Single Syringe Pump, NSW, Australia) in the absence or presence of anticoagulant. Carotid blood flow was assessed for 60 minutes after the start of treatment. Upon completion of the intravenous infusion, the jugular catheter was removed and the proximal (toward the heart) clamp was tightened to prevent blood loss. After thrombolytic therapy, recovery of carotid blood flow was assessed using mean blood flow recorded by LabChart software (version 7.0, ADInstruments, NSW, Australia). Recanalization was identified by the return of blood flow and was classified as stable, unstable, transient, or not restored. No recovery represented persistent occlusion for the period of monitoring without a recanalization event; transient blood flow was characterized by a transient recanalization event followed by reocclusion; fluctuations in mean blood flow without complete vessel reocclusion were defined as unstable blood flow; and stable blood flow was characterized as blood flow without reocclusion or fluctuations in mean blood flow.

[0216] We investigated the ability of the peptide of SEQ ID NO:1 in combination with rtPA therapy to recanalize occluded blood vessels in an experimental thrombolysis assay. A schematic of the experimental thrombolysis timeline is shown in FIG. 5A along with images of typical injuries including clot formation shown in FIG. 5B-E. FIG. 6 shows the results obtained with no treatment (A), rtPA therapy alone (B), rtPA therapy in combination with hirudin (C) and rtPA therapy in combination with MDL-2 (D). These results show that blood vessels remain occluded in the absence of treatment and with rtPA therapy alone. rtPA therapy in the presence of MDL-2 results in a significant increase in blood flow compared to hirudin on a molar equivalent basis.

Claims

1. A peptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:

2.

2. 2. The peptide of claim 1, wherein at least one residue of SEQ ID NO:1 or SEQ ID NO:2 is a sulfated tyrosine residue.

3. 3. The peptide of claim 1, wherein the tyrosine at position 32 and the tyrosine at position 35 in SEQ ID NO:1 are sulfated.

4. 3. The peptide of claim 1, wherein the tyrosine at position 33 and the tyrosine at position 36 in SEQ ID NO:2 are sulfated.

5. The peptide according to any one of claims 1 to 4, which consists of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

6. Formula 1: A-B (In the formula, A is a peptide according to any one of claims 1 to 6; B is a peptide having a sequence shown in one of SEQ ID NOs: 3 to 5. Fusion peptide.

7. A peptide according to any one of claims 1 to 6, which at least partially inhibits thrombin.

8. 8. The peptide of claim 7, which inhibits thrombin with an inhibition constant Ki of less than about 500 pM and comprises an amino acid sequence as set forth in SEQ ID NO:1, wherein the tyrosine at position 32 and the tyrosine at position 35 in SEQ ID NO:1 are sulfated.

9. 9. The peptide of any one of claims 1 to 8, which reduces clotting time by at least 20% based on molar equivalents of the peptide and known thrombin inhibitors compared to known thrombin inhibitors that result in significant blood loss.

10. 10. The peptide of any one of claims 1 to 9, comprising a clotting time of about 60 seconds to about 200 seconds at a concentration of greater than about 4 μg / mL, as measured using an in vitro activated partial thromboplastin time (aPTT) assay.

11. 11. The peptide of any one of claims 1 to 10, which when administered to an individual in an amount of from about 1 μg per kg of said individual to about 10 mg per kg of said individual, results in a clotting time of from about 60 seconds to about 200 seconds, as measured ex vivo using an in vitro activated partial thromboplastin time (aPTT) assay.

12. A peptide according to any one of claims 1 to 11, which does not result in significant blood loss.

13. 13. The peptide of any one of claims 1 to 12, which reduces blood loss by at least 10-fold based on molar equivalents of the peptide and the known thrombin inhibitor, as compared to known thrombin inhibitors that result in significant blood loss.

14. 14. The peptide of claim 9 or 13, wherein the known thrombin inhibitor is hirudin.

15. A pharmaceutical composition comprising a peptide according to any one of claims 1 to 14 together with a pharma- ceutically effective carrier, diluent or excipient.

16. A polynucleotide comprising a nucleotide sequence encoding the peptide according to any one of claims 1 to 14.

17. A vector or expression construct comprising the polynucleotide of claim 16.

18. 18. A cell comprising a polynucleotide according to claim 16 or a vector or construct according to claim 17.

19. The cell of claim 18, which comprises a protein tyrosine sulfotransferase that enables sulfation of tyrosine residues.

20. A method for preventing or treating a thromboembolic condition in an individual in need thereof, comprising, consisting essentially of or consisting of administering to said individual a therapeutically effective amount of a peptide according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15, thereby preventing or treating said thromboembolic condition in said individual.

21. A method of treating a thromboembolic condition or reducing the severity of a symptom of a thromboembolic condition in an individual comprising, consisting essentially of, or consisting of administering to said individual in need thereof a therapeutically effective amount of a peptide according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15, thereby treating said thromboembolic condition or reducing the severity of said symptoms of said thromboembolic condition in said individual.

22. Use of a peptide according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15 in the manufacture of a medicament for the prevention or treatment of a thromboembolic condition in an individual in need thereof.

23. A peptide according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15 for use in the prevention or treatment of a thromboembolic condition in an individual in need thereof.

24. 24. The method according to claim 20 or 21, the use according to claim 22 or the peptide according to claim 23, wherein the thromboembolic condition is selected from myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, disseminated intravascular coagulation, cardiovascular and cerebrovascular thrombosis, thrombosis associated with postoperative trauma, obesity, pregnancy, side effects of oral contraceptives, hypercoagulable states associated with prolonged immobility and hematological, immune or rheumatic disorders, unstable angina, arteriosclerosis, reocclusion of a vessel after angioplasty with a balloon catheter or blood clotting in hemodialysis.

25. 23. The method, use or peptide of claim 22, wherein said thromboembolic condition is stroke.

26. A method for inhibiting or minimizing the progression of blood clot formation associated with inflammation caused by a viral infection in an individual, comprising, consisting essentially of, or consisting of administering a therapeutically effective amount of a peptide according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15 to an individual experiencing inflammation caused by a viral infection, thereby inhibiting or minimizing the progression of blood clot formation in said individual. method.

27. The method according to any one of claims 20, 21 or 24-26, the use according to any one of claims 22, 24 or 25 or the peptide according to any one of claims 23-25, wherein the peptide is administered in combination with one or more thrombolytic agents, one or more platelet inhibitors or a combination thereof.

28. The method according to any one of claims 20, 21 or 24-27, the use according to any one of claims 22, 24, 25 or 27 or the peptide according to any one of claims 23-25 ​​or 27, wherein the peptide is administered by bolus injection or intravenous infusion.

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